產(chǎn)品詳情

  • 產(chǎn)品名稱：TFPI ELISA Kit(TissueFactorPathwayInhibitor(Lipoprotein-AssociatedCoagulationInhi

  • 產(chǎn)品型號(hào)：
  • 產(chǎn)品廠商：國(guó)內(nèi)供應(yīng)3
  • 產(chǎn)品文檔：
你添加了1件商品 查看購(gòu)物車
簡(jiǎn)單介紹：
TFPI ELISA Kit(TissueFactorPathwayInhibitor(Lipoprotein-AssociatedCoagulationInhibitor))
詳情介紹：
Purpose This immunoassay kit allows for the specific measurement of human Tissue Factor Pathway Inhibitor (TFPI) concentrations in plasma and cell culture supernates.
Sample Type Plasma, Cell Culture Supernatant
Analytical Method Quantitative
Detection Method Colorimetric
Specificity This assay recognizes recombinant and natural human TFPI.
Cross-Reactivity (Details) No significant cross-reactivity or interference was observed.
Characteristics Homo sapiens,Human,Tissue factor pathway inhibitor,TFPI,Extrinsic pathway inhibitor,EPI,Lipoprotein-associated coagulation inhibitor,LACI,TFPI,LACI,TFPI1
Components Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1x120μl), Detection Reagent B (1x120μl), Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)
Alternative Name TFPI (TFPI ELISA Kit Abstract)
Background In plasma, Tissue Factor Pathway Inhibitor (TFPI) is found in several forms: one at 36,000 kD, one at 43,000 kD, and the various truncated forms. This size heterogeneity appears in part to be the result of the formation of mixed disulfide complexes between TFPI and apolipoprotein AII. Approximately 10% of total TFPI is carried by platelets, which release TFPI once they are activated by thrombin. Thus, at the site of a wound, where platelets aggregate, elevated levels of TFPI are present. TFPI has dual inhibitory function (against factor Xa and factor VIIa/TF). This is consistent with the presence of multiple Kunitz-type domains: (Kunitz for factor VIIa/TF and Kunitz for factor Xa interaction). Based on the initial isolation of the inhibitor, it was found that TFPI inhibits factor VIIa/TF and directly inhibits factor Xa by binding at or near its serine active site. The mechanism of action for factor Xa dependent inhibition of factor VIIa/TF by TFPI involves the formation of a quaternary factor Xa/TFPI/factor VIIa/TF complex. This results from the initial binding of factor Xa to TFPI, with subsequent binding of the factor Xa-TFPI complex to factor VIIa/TF. Alternatively, TFPI might bind to the factor Xa-VIIa/TF complex. Quantitation of TFPI, with its dual inhibitory role against factor Xa and factor VIIa/TF, offers insight into the mechanism of disseminated intravascular coagulation (DIC) triggered by tissue factor. It has been observed that infusion of TFPI abrogates DIC and prevents reocclusion following thrombolysis induced by injury. Clinical conditions in which thrombosis appears to be initiated by TF and in which TFPI treatment may be potentially useful include sepsis, inflammatory disease and transplant rejection. TFPI has potential as an antithrombotic agent.
Sample Volume 100 μL
Plate Pre-coated
Protocol This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TFPI has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TFPI present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for TFPI is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of TFPI bound in the initial step. The color development is stopped and the intensity of the color is measured.
Reagent Preparation

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 20000 pg/mL. Allow the standard to sit for a minimum of 15 minutes 3 with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (20000 pg/mL). The Sample Diluent serves as the zero standard (0 pg/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.
Sample Collection Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Plasma - Blood should be collected in a tube containing 3.2% buffered sodium citrate. Evacuated collection tubes must be filled to completion to ensure a proper blood to anticoagulant ratio. The sample should be mixed immediately by gentle inversion at least six times to ensure adequate mixing of the anticoagulant with the blood. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.
Assay Procedure

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C . Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:
1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. 4
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
Calculation of Results

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the TFPI concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Restrictions For Research Use only
Handling Advice 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Storage 4 °C/-20 °C
Storage Comment The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.